The effect of charge density on the velocity and attenuation of ultrasound waves in human cancellous bone

Cancellous bone is a highly porous material, and two types of waves, fast and slow, are observed when ultrasound is used for detecting bone diseases. There are several possible stimuli for bone remodelling processes, including bone fluid flow, streaming potential, and piezoelectricity. Poroelasticity has been widely used for elucidating the bone fluid flow phenomenon, but the combination of poroelasticity with charge density has not been introduced. Theoretically, general poroelasticity with a varying charge density is employed for determining the relationship between wave velocity and attenuation with charge density. Fast wave velocity and attenuation are affected by porosity as well as charge density; however, for a slow wave, both slow wave velocity and attenuation are not as sensitive to the effect of charge density as they are for a fast wave. Thus, employing human femoral data, we conclude that charged ions gather on trabecular struts, and the fast wave, which moves along the trabecular struts, is significantly affected by charge density.     

Interstitial fluid flow in the osteon with spatial gradients of mechanical properties: a finite element study

Bone remodelling is the process that maintains bone structure and strength through adaptation of bone tissue mechanical properties to applied loads. Bone can be modelled as a porous deformable material whose pores are filled with cells, organic material and interstitial fluid. Fluid flow is believed to play a role in the mechanotransduction of signals for bone remodelling. In this work, an osteon, the elementary unit of cortical bone, is idealized as a hollow cylinder made of a deformable porous matrix saturated with an interstitial fluid. We use Biot’s poroelasticity theory to model the mechanical behaviour of bone tissue taking into account transverse isotropic mechanical properties. A finite element poroelastic model is developed in the COMSOL Multiphysics software. Elasticity equations and Darcy’s law are implemented in this software; they are coupled through the introduction of an interaction term to obtain poroelasticity equations. Using numerical simulations, the investigation of the effect of spatial gradients of permeability or Poisson’s ratio is performed. Results are discussed for their implication on fluid flow in osteons: (i) a permeability gradient affects more the fluid pressure than the velocity profile; (ii) focusing on the fluid flow, the key element of loading is the strain rate; (iii) a Poisson’s ratio gradient affects both fluid pressure and fluid velocity. The influence of textural and mechanical properties of bone on mechanotransduction signals for bone remodelling is also discussed.     

Fluid pressure relaxation depends upon osteonal microstructure: modeling an oscillatory bending experiment.

When bone is mechanically loaded, bone fluid flow induces shear stresses on bone cells that have been proposed to be involved in bone's mechanosensory system. To investigate bone fluid flow and strain-generated potentials, several theoretical models have been proposed to mimic oscillatory four-point bending experiments performed on thin bone specimens. While these previous models assume that the bone fluid relaxes across the specimen thickness, we hypothesize that the bone fluid relaxes primarily through the vascular porosity (osteonal canals) instead and develop a new poroelastic model that integrates the microstructural details of the lacunar-canalicular porosity, osteonal canals, and the osteonal cement lines. Local fluid pressure profiles are obtained from the model, and we find two different fluid relaxation behaviors in the bone specimen, depending on its microstructure: one associated with the connected osteonal canal system, through which bone fluid relaxes to the nearby osteonal canals; and one associated with the thickness of a homogeneous porous bone specimen (approximately 1 mm in our model), through which bone fluid relaxes between the external surfaces of the bone specimen at relatively lower loading frequencies. Our results suggest that in osteonal bone specimens the fluid pressure response to cyclic loading is not sensitive to the permeability of the osteonal cement lines, while it is sensitive to the applied loading frequency. Our results also reveal that the fluid pressure gradients near the osteonal canals (and thus the fluid shear stresses acting on the nearby osteocytes) are significantly amplified at higher loading frequencies.     

Lung, heart, and kidney express high levels of mRNA for the vitamin K-dependent matrix Gla protein. Implications for the possible functions of matrix Gla protein and for the tissue distribution of the gamma-carboxylase

We have used cDNA probes for two small vitamin K-dependent bone matrix proteins, bone Gla protein (BGP) and matrix Gla protein (MGP), to evaluate the possibility that either of these proteins might be synthesized by the various soft tissues previously shown to have gamma-carboxylase activity. BGP mRNA was found in bone but not in any of the soft tissues tested, a result which reinforces the view that plasma BGP is a specific marker for bone metabolism. In contrast, MGP mRNA was found in all rat tissues examined. Lung and heart have 10-fold higher levels of MGP mRNA than bone, and kidney has a 5-fold higher level. Despite the high levels of MGP mRNA in heart and kidney, these tissues contain 40-500-fold lower concentrations of MGP protein than bone. Immunofluorescence was used to identify cells that contain MGP in kidney, lung, heart, and spleen. In each tissue, MGP was found in discrete tissue-specific cell types. In most of the soft tissues tested, MGP is the first well characterized substrate for the vitamin K-dependent carboxylase found to be synthesized. The exceptionally broad tissue distribution for MGP synthesis demonstrates that the function of MGP is not specific to connective tissues, and the low levels of MGP antigen in soft tissues with high MGP mRNA levels indicate that MGP is unlikely to act solely by virtue of its accumulation in an extracellular matrix.     

The strain-generated electrical potential in cartilaginous tissues: a role for piezoelectricity

The strain-generated potential (SGP) is a well-established mechanism in cartilaginous tissues whereby mechanical forces generate electrical potentials. In articular cartilage (AC) and the intervertebral disc (IVD), studies on the SGP have focused on fluid- and ionic-driven effects, namely Donnan, diffusion and streaming potentials. However, recent evidence has indicated a direct coupling between strain and electrical potential. Piezoelectricity is one such mechanism whereby deformation of most biological structures, like collagen, can directly generate an electrical potential. In this review, the SGP in AC and the IVD will be revisited in light of piezoelectricity and mechanotransduction. While the evidence base for physiologically significant piezoelectric responses in tissue is lacking, difficulties in quantifying the physiological response and imperfect measurement techniques may have underestimated the property. Hindering our understanding of the SGP further, numerical models to-date have negated ferroelectric effects in the SGP and have utilised classic Donnan theory that, as evidence argues, may be oversimplified. Moreover, changes in the SGP with degeneration due to an altered extracellular matrix (ECM) indicate that the significance of ionic-driven mechanisms may diminish relative to the piezoelectric response. The SGP, and these mechanisms behind it, are finally discussed in relation to the cell response.     

Fluid-induced osteolysis: modelling and experiments

A model to calculate bone resorption driven by fluid flow at the bone–soft tissue interface is developed and used as a basis for computer calculations, which are compared to experiments where bone is subjected to fluid flow in a rat model. Previous models for bone remodelling calculations have been based on the state of stress, strain or energy density of the bone tissue as the stimulus for remodelling. We believe that there is experimental support for an additional pathway where an increase in the amount of the cells directly involved in bone removal, the osteoclasts, is caused by fluid pressure, flow velocity or other parameters related to fluid flow at the bone–soft tissue interface, resulting in bone resorption.     

Poroelastic analysis of interstitial fluid flow in a single lamellar trabecula subjected to cyclic loading

Trabecula, an anatomical unit of the cancellous bone, is a porous material that consists of a lamellar bone matrix and interstitial fluid in a lacuno-canalicular porosity. The flow of interstitial fluid caused by deformation of the bone matrix is believed to initiate a mechanical response in osteocytes for bone remodeling. In order to clarify the effect of the lamellar structure of the bone matrix—i.e., variations in material properties—on the fluid flow stimuli to osteocytes embedded in trabeculae, we investigated the mechanical behavior of an individual trabecula subjected to cyclic loading based on poroelasticity. We focused on variations in the trabecular permeability and developed an analytical solution containing both transient and steady-state responses for interstitial fluid pressure in a single trabecular model represented by a multilayered two-dimensional poroelastic slab. Based on the obtained solution, we calculated the pressure and seepage velocity of the interstitial fluid in lacuno-canalicular porosity, within the single trabecula, under various permeability distributions. Poroelastic analysis showed that a heterogeneous distribution of permeability produces remarkable variations in the fluid pressure and seepage velocity in the cross section of the individual trabecula, and suggests that fluid flow stimuli to osteocytes are mostly governed by the value of permeability in the neighborhood of the trabecular surfaces if there is no difference in the average permeability in a single trabecula.     

理解生物组织细胞力学信号传导机制的必要分析：以关节软骨为例

关节软骨是覆盖于骨关节面的一薄层低摩擦、耐磨损、负重的水化组织。这种功能通过散在镶嵌于软骨组织深层致密胞外组织内的软骨细胞的代谢和生物合成作用来维持。这种代谢和生物合成进程很大一部分是由物理因素,如应力、张力、电流及电势、液压及渗透压等来调控的。这两者都存在于这一带电的、渗透性的基质当中。本文主要讲述关节软骨及软骨细胞的机械一电化学行为及理论模型的最近进展,同时着眼于软骨细胞生物合成活动的物理调控及其对于组织维持、功能组织工程软骨修复及再生医学的意义。     

A finite element analysis of the indentation stress-relaxation response of linear biphasic articular cartilage.

The indentation problem of a thin layer of hydrated soft tissue such as cartilage or meniscus by a circular plane-ended indenter is investigated. The tissue is represented by a biphasic continuum model consisting of a solid phase (collagen and proteoglycan) and a fluid phase (interstitial water). A finite element formulation of the linear biphasic continuum equations is used to solve an axisymmetric approximation of the indentation problem. We consider stress-relaxation problems for which analytic solution is intractable; where the indenter is impermeable (solid) and/or when the interface between the indenter and tissue is perfectly adhesive. Thicknesses corresponding to a thin and thick specimen are considered to examine the effects of tissue thickness. The different flow, pressure, stress and strain fields which are predicted within the tissue, over time periods typically used in the mechanical testing of soft tissues, will be presented. Results are compared with the case of a porous free-draining indenter with a perfectly lubricated tissue-indenter interface, for which an analytic solution is available, to show the effects of friction at the tissue-indenter interface, and the effects of an impermeable indenter. While these effects are present for both thin and thick tissues, they are shown to be more significant for the thin tissue. We also examine the effects of the stiffness of the subchondral bone on the response of the soft tissue and demonstrate that the subchondral bone substrate can be modeled as a rigid, impermeable boundary. The effects of a curved tissue-subchondral bone interface, and the early time response are also studied. For physiologically reasonable levels of curvature, we will show that the curved tissue-subchrondal bone interface has negligible influence on the tissue response away from the interface. In addition, the short-time stress-relaxation responses of the tissue (e.g., at times less than 1s) demonstrate the essential role of the fluid phase in supporting the load applied to the tissue, and by extrapolation to shorter times characteristics of normal joint motion, suggest the essential role of a biphasic model in representing soft tissue behavior in joint response.     

Guarded burr for deepening of nasofrontal junction

Appropriate deepening of the nasofrontal junction remains one of the most difficult parts of rhinoplasty. A guarded burr is introduced that provides a safe and effective means of deepening or creating a shallow nasofrontal groove. The burr has a special guard that prevents the cutting of soft tissue. The guarded burr is introduced in position through the routine elevation of the bridge soft tissue subperiosteally. The safety latch is used for orientation. With gentle movement along the line connecting the canthi, the desired amount of bone can be removed from the nasofrontal junction with little time consumed. It is important to avoid continuous friction, since this might result in significant heat production and thereby thermal injury to the soft tissue and fluid collection.     

The speed of sound through trabecular bone predicted by Biot theory

Cancellous bone is a highly porous material filled with fluid. The mechanical properties of cancellous bone determine whether the bone is normal or osteoporotic. Wave propagation can be used to measure the elastic constants of cancellous bone. Recently, poroelasticity theory has been used to predict the elastic constants of cancellous bone from the wave velocities. In this study, it is shown that the fast wave, predicted by the Biot theory, corresponds to the wave penetrating the trabeculae, while the slow wave is determined by the interaction between the trabeculae and the fluid. The trabecular shape does not affect the wave velocity significantly when using the variable, which is determined by the microstructure, and the slow wave velocity decreases after the porosity reaches 80%.     

Streaming potential of intact wet bone

The conversion of mechanical loads to bioelectrical signals in bone have been suggested to control repair and remodeling. These signals in wet bone are attributed to the electrokinetic behavior where mechanical forces cause electrical signals due to motion of an ion carrying extracellular fluid in the bone matrix (streaming potentials). Streaming potential experiments were performed on control and chemically treated intact wet bone plugs in aphosphate and phosphate buffers to examine the contribution of bone constituents to the electrokinetic behavior of bone tissue. Data indicate that the organic constituents of bone dominate streaming potentials. Slopes of streaming potential vs pressure are related to the electrokinetic (zeta) potential. The slopes should be analyzed in the low pressure region where data is mainly linear. Comparisons of estimated zeta potentials from streaming potentials with existing data obtained by particle electrophoresis showed similar trends.     

Tendon exhibits complex poroelastic behavior at the nanoscale as revealed by high-frequency AFM-based rheology

Tendons transmit load from muscle to bone by utilizing their unique static and viscoelastic tensile properties. These properties are highly dependent on the composition and structure of the tissue matrix, including the collagen I hierarchy, proteoglycans, and water. While the role of matrix constituents in the tensile response has been studied, their role in compression, particularly in matrix pressurization via regulation of fluid flow, is not well understood. Injured or diseased tendons and tendon regions that naturally experience compression are known to have alterations in glycosaminoglycan content, which could modulate fluid flow and ultimately mechanical function. While recent theoretical studies have predicted tendon mechanics using poroelastic theory, no experimental data have directly demonstrated such behavior. In this study, we use high-bandwidth AFM-based rheology to determine the dynamic response of tendons to compressive loading at the nanoscale and to determine the presence of poroelastic behavior. Tendons are found to have significant characteristic dynamic relaxation behavior occurring at both low and high frequencies. Classic poroelastic behavior is observed, although we hypothesize that the full dynamic response is caused by a combination of flow-dependent poroelasticity as well as flow-independent viscoelasticity. Tendons also demonstrate regional dependence in their dynamic response, particularly near the junction of tendon and bone, suggesting that the structural and compositional heterogeneity in tendon may be responsible for regional poroelastic behavior. Overall, these experiments provide the foundation for understanding fluid-flow-dependent poroelastic mechanics of tendon, and the methodology is valuable for assessing changes in tendon matrix compressive behavior at the nanoscale.     

Fibroblast alignment under interstitial fluid flow using a novel 3-D tissue culture model

Interstitial flow is an important component of the microcirculation and interstitial environment, yet its effects on cell organization and tissue architecture are poorly understood, in part due to the lack of in vitro models. To examine the effects of interstitial flow on cell morphology and matrix remodeling, we developed a tissue culture model that physically supports soft tissue cultures and allows microscopic visualization of cells within the three-dimensional matrix. In addition, pressure-flow relationships can be continuously monitored to evaluate the bulk hydraulic resistance as an indicator of changes in the overall matrix integrity. We observed that cells such as human dermal fibroblasts aligned perpendicular to the direction of interstitial flow. In contrast, fibroblasts in static three-dimensional controls remained randomly oriented, whereas cells subjected to fluid shear as a two-dimensional monolayer regressed. Also, the dynamic measurements of hydraulic conductivity suggest reorganization toward a steady state. These primary findings help establish the importance of interstitial flow on the biology of tissue organization and interstitial fluid balance.     

Improved characterization of cartilage mechanical properties using a combination of stress relaxation and creep

Mechanical characterization of cartilage, other soft tissues and gels has become a ubiquitous and essential aspect of biomechanics and biomaterials research. Current progress in theoretical modeling and tools for data analysis often exceed what is required for routine mechanical characterization assays in experimental studies, making selection of methodologies difficult for the nonspecialist. We have therefore developed an approach for measurement of confined compression modulus and hydraulic permeability based on simple poroelasticity theory and requiring only linear regression tools for data analysis. This technique involves a new application of an early-time solution for creep combined with stress relaxation measurements to characterize soft tissue mechanical parameters as a function of compressive strain or water content. This combined methodology allows measurement of hydraulic permeability by two different techniques with only a modest increase in experimental duration, providing a more precise assessment of permeability and associated measurement error.     

Therapeutic effects of electromagnetic fields in the stimulation of connective tissue repair

The therapeutic effects of electric and magnetic fields have been studied largely for their promotion of connective tissue repair. The most widely studied application concerns bone repair and deals with acceleration of the healing of fresh fractures, delayed and non-unions, incorporation of bone grafts, osteoporosis, and osteonecrosis. More recently the effects of these fields upon the repair of cartilage and soft fibrous tissues have been described. In all these experimental systems and clinical applications an acceleration of extracellular matrix synthesis and tissue healing has been observed. A degree of specificity, in terms of the parameters of applied energy and biological response, is hypothesized.     

Cabozantinib Inhibits Growth of Androgen-Sensitive and Castration-Resistant Prostate Cancer and Affects Bone Remodeling

Cabozantinib is an inhibitor of multiple receptor tyrosine kinases, including MET and VEGFR2. In a phase II clinical trial in advanced prostate cancer (PCa), cabozantinib treatment improved bone scans in 68% of evaluable patients. Our studies aimed to determine the expression of cabozantinib targets during PCa progression and to evaluate its efficacy in hormone-sensitive and castration-resistant PCa in preclinical models while delineating its effects on tumor and bone. Using immunohistochemistry and tissue microarrays containing normal prostate, primary PCa, and soft tissue and bone metastases, our data show that levels of MET, P-MET, and VEGFR2 are increasing during PCa progression. Our data also show that the expression of cabozantinib targets are particularly pronounced in bone metastases. To evaluate cabozantinib efficacy on PCa growth in the bone environment and in soft tissues we used androgen-sensitive LuCaP 23.1 and castration-resistant C4-2B PCa tumors. In vivo, cabozantinib inhibited the growth of PCa in bone as well as growth of subcutaneous tumors. Furthermore, cabozantinib treatment attenuated the bone response to the tumor and resulted in increased normal bone volume. In summary, the expression pattern of cabozantinib targets in primary and castration-resistant metastatic PCa, and its efficacy in two different models of PCa suggest that this agent has a strong potential for the effective treatment of PCa at different stages of the disease.     

Immunohistochemical localization of bone sialoprotein in foetal porcine bone tissues: comparisons with secreted phosphoprotein 1 (SPP-1, osteopontin) and SPARC (osteonectin)

Summary Bone sialoprotein (BSP) is a prominent component of bone tissues that is expressed by differentiated osteoblastic cells. Affinity-purified antibodies to BSP were prepared and used in combination with biotin-conjugated peroxidase-labeled second antibodies to demonstrate the distribution of this protein in sections of demineralized foetal porcine tibia and calvarial bone. Staining for BSP was observed in the matrix of mineralized bone and also in the mineralized cartilage and associated cells of the epiphysis, but was not observed in the hypertrophic zone nor in any of the soft tissues including the periosteum. In comparison, SPP-1 (osteopontin) and SPARC (osteonectin), which are also major proteins in porcine bone, were observed in the cartilage as well as in the mineralized bone matrix, In addition, SPARC was also present in soft connective tissues. Although SPP-1 distribution was more restricted than SPARC, hypertrophic chondrocytes, periosteal cells and some stromal cells in the bone marrow spaces were stained in addition to osteoblastic cells. The variations in the distribution and cellular expression of BSP, SPARC and SPP-1 in bone and mineralizing cartilage indicate these proteins perform different functions in the formation and remodelling of mineralized connective tissues.     

Matrix metalloproteinases and TIMPs: properties and implications for the rheumatic diseases

The matrix metalloproteinases (MMPs) are a unique family of metalloenzymes, which, once activated, can destroy all the components of cartilage. MMPs are found in resorbing cartilage, bone, rheumatoid and osteoarthritic synovial fluid, and adjacent soft tissues. The active enzymes are all inhibited by tissue inhibitors of metalloproteinases (TIMPs). The relative amounts of active MMPs and TIMPs are important in determining whether cartilage is broken down in joint diseases. Conventional treatments for arthritis do little to affect the underlying joint destruction, but new drugs are now available that can specifically block active MMPs. These potent inhibitors prevent the destruction of cartilage both in vitro and in animal models of arthritis. Future trials in patients will test their effectiveness in the prevention of cartilage destruction.